Biological fluid assay system

ABSTRACT

A biological fluid assay system and method for the determination of immunoreactive characteristics of biological specimen and more particularly for the qualitative determination of immunological reactions. The apparatus includes a centrifuge rotor, a disposable belt mountable on the centrifuge rotor, and a plurality of light transmissive chambers as components of the removable belt, each of the chambers having a vertical apex and a horizontal radial apex for accepting a sample comprising a specimen and an appropriate reagent. An illumination system projects an image of the sample while a linear photosensitive array detects the image of the sample for measuring the vertical dimension of the sample and a microprocessor analyzes the vertical dimension of the sample. A sample is radially accelerated to compress the particulate portion of the sample or reagent into a compact mass in the extreme radial portion of the transparent chamber. The centrifugal force on the sample is balanced with the gravitational force acting on the compact mass by decelerating the rotor. The vertical dimension of the compact mass is measured while the sample is rotating. The vertical dimension of the compact mass is again measured after a delay to determine the presence of vertical streaming. The difference between the vertical dimension of the compact mass before streaming and the vertical dimension of the mass after streaming is completed, due to gravitational attraction, is determined.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of prior application Ser. No. 546,345,filed Oct. 28, 1983.

FIELD OF THE INVENTION

The present invention relates to the determination of the reactivecharacteristics of fluid specimens and more particularly relates to amethod and automated apparatus for the qualitative determination ofimmunological reactions of biological specimens.

BACKGROUND OF THE INVENTION

The microscope slide technique for manually performing blood groupingtests is not sensitive to subtle reactions and requires strict attentionto protocol to avoid the possibility of sample misidentification. As analternative to the microscope slide technique the test tube centrifugetechnique has been utilized. Additionally, the test tube centrifugetechnique has been used to augment or to confirm the microscope slidetechnique procedures. The test tube centrifuge technique has thecharacteristic of being more sensitive to subtle reactions than themicroscope slide technique. However, the test tube centrifuge techniqueis more labor intensive, is subject to interpretation by the laboratorytechnician and has the inherent problem of assuring accurate sampleidentification.

The "microwell" test method has been developed to acquire the maximumutilization of the materials involved in blood grouping and tostandardize the interpretation and the evaluation of biologicalspecimens to provide more consistent results. The "microwell" testmethod uses a plate containing numerous small centrifuge tubes or wells.

Automated reading techniques for blood grouping have typically beenlimited to the measurement of turbidity. Turbidity results from thebreakup of centrifuged red blood cells. A method of initiating thedissociation of red blood cells is to mix the cells with a reagent andto subject the mixture to vibration. The dissociation of the red cellsis indicative of a negative reaction between red blood cells and theadded reagent. If a positive reaction occurs after being subjected tovibration, the red cells remain tightly associated. The cohesiveattraction of the red cells when subjected to the reagent is anindication of a positive reaction. Typically, a positive reactionbetween the red blood cells and the reagent yields a compact mass of redblood cells and a clear supernatant.

Turbidity may be measured based upon the opacity of the specimen. Alight or energy beam is transmitted through the specimen. A detectordetermines the reduction in the intensity of the light or energy beamcaused by the scattering or the absorption of the beam by the specimen.The turbidity of the specimen is measured as a function of the reductionin transmitted light or energy. Specifically, the reduction of theintensity of the transmitted beam is caused by the loss of light orenergy due to the scattering and the absorption of the incident beam bythe suspended red cells. The number of available suspended red cells isdirectly proportional to the amount of dissociation of red cells causedby negative reactions.

Despite important advances in the prior art, there is still a need foran automated blood grouping system and a method which reduces thesubjective interpretation of reactions by laboratory personnel, which iseasily used, which requires little technical expertise and fewpersonnel, and which is comparatively inexpensive.

SUMMARY OF INVENTION

Recognizing the need for an improved system and method for automaticallydetermining blood groups, it is, therefore, a general attribute of thepresent invention to provide a novel automated blood grouping system andmethod.

A feature of the present invention is to provide a novel automated bloodgrouping method and system which positively identifies the specimen fromeach patient.

Another feature of the present invention is to provide a novel apparatuswhich performs laboratory procedures, normally accomplished manually, inan automatic, completely reproducible sequence, assuring consistentresults.

Yet another feature of the present invention is to provide a novelautomated blood grouping method which may be performed in rapidsuccession with an extremely high degree of accuracy.

Still another feature of the present invention is to provide a novelautomated blood grouping method and system that accurately determinesthe biological parameters of blood specimen based upon an accurateevaluation of the reaction, or lack thereof, between a biologicalspecimen and a reagent.

Additional features and advantages of the invention will be set forth inpart in the description which follows and in part will become apparentfrom the description, or may be learned by practice of the invention.The features and advantages of the invention may be realized andobtained by means of instrumentalities, combinations and stepsparticularly pointed out in the appended claims.

In accordance with one embodiment of the present invention a method fordetermining the reactive characteristics of a biological fluid specimenbegins with the step of preparing a sample including the specimen and anappropriate reagent such that the sample is at least partially opaque.The sample is centrifuged and then a first measurement is made of alinear dimension of an opaque portion of the sample. Thereafter, asubsequent measurement of the same linear dimension of the sample ismade. Then the first measured dimension is compared to the secondmeasured dimension.

In accordance with another embodiment of the present invention anapparatus for determining the reactive characteristics of a biologicalfluid specimen includes a centrifuge rotor and an energy transmissive,flexible belt formable into a cylinder and removably mountable formovement in unison with the rotor. The belt includes means for acceptinga plurality of specimens. A detection system irradiates the plurality ofspecimens contained within said belt and optically measures the reactivecharacteristics of the specimens.

In further summary, it should be understood that blood groupingaccording to the present invention is accordingly, relates broadly to amethod and an apparatus for differentiating between positive andnegative immunological reactions indicated by the affinity of particlesassociated with a biological specimen and an associated reagent ofinterest. The present invention also has application in otherimmunoassays, such as determinations of hepatitis, rheumatoid arthritis,and infectious mononucleosis, where opaque particles other than humanred blood cells are used as carriers for immunological agents(antigens). Suitable opaque particles include barium sulphate, latex,animal red blood cells, or any particle to which an antigen of interestmay be attached and viewed.

BRIEF DESCRIPTION OF THE DRAWING

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate a preferred embodiment of theinvention and, together with the general description of the inventiongiven above and the detailed description of the preferred embodimentgiven below, serve to explain the principles of the invention.

FIG. 1 is a perspective view of one embodiment of the automated bloodgrouping system of the present invention;

FIG. 2 is a cross-sectional view taken generally along section line 2--2in FIG. 1

FIG. 3 is a cross-sectional view taken along section line 3--3 in FIG. 2

FIG. 4 is an enlarged plan view taken generally along section line 4--4in FIG. 3;

FIG. 5 is a cross-sectional view of the present invention takengenerally along section line 5--5 of FIG. 4;

FIG. 6 is an enlarged elevational view of a segment of a disposable beltshown in FIG. 4;

FIG. 7 is a plan view taken along the line 7--7 of FIG. 6;

FIG. 8 is a vertical, cross-sectional view taken generally along theline 8--8 of FIG. 6;

FIGS. 9 are vertical cross-sectional views taken generally along theline 9--9 in FIG. 6 at different stages during the use of the presentinvention illustrating the positions of the sample, compacted red bloodcells, supernatant and streaming red blood cells;

FIG. 10 is an enlarged partial plan view taken generally along thesection line 10--10 of FIG. 2;

FIG. 11 is a section view taken generally along the curved arc 11--11 inFIG. 9;

FIG. 12 is a cross-sectional view taken generally along the section line12--12 in FIG. 11;

FIG. 13 is an enlarged, cross-sectional, cut-away view illustrating thereagent reservoirs of the reagent dispensing device taken along thesection line 13--13 in FIG. 2;

FIG. 14 is a partial cross-sectional view illustrating a reagentreservoir of the present invention taken along the section line 14--14in FIG. 13;

FIG. 15 is a schematic diagram illustrating the microprocessor system ofthe present invention;

FIG. 16 is a schematic diagram of one embodiment of a first Input/OutputBoard and the associated peripherals as practiced in one embodiment ofthe present invention;

FIG. 17 is a partial schematic diagram, continued in FIG. 18, of oneembodiment of the power driver circuitry and the associated peripheralsas practiced in one embodiment of the present invention;

FIG. 18 is a partial schematic diagram, continued from FIG. 17, of oneembodiment of the second motor driver circuitry and the associatedperipherals as practiced in the present invention;

FIG. 19 is a flow chart of one embodiment of the present invention; and

FIG. 20 is a flow chart of one embodiment for preparing the samples forpracticing the present invention

The above general description and the following detailed description aremerely illustrative of the generic invention, and additional modes,advantages and particulars of this invention will be readily suggestedto those skilled in the art without departing from the spirit and scopeof the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring to the drawing wherein like reference characters are used forlike parts throughout the several views, a biological fluid assayapparatus 10, shown in FIGS. 1 and 2, includes a centrifuge rotor 300, asingle use belt 400 mounted on the rotor 300, a microprocessor 600, anoptical system 500, a pipetter-diluter device 100 and a reagentdispensing device 200. The biological fluid assay apparatus 10 alsoincludes a housing 11 having a top structure 12 with a planar worksurface 20, on which various controls are located, and a bottomstructure 14. The biological fluid assay apparatus 10 is electricallyactivated by a switch 602 on the front vertical side 22 of the topstructure 12. The operator utilizes a keyboard 604 to input theappropriate test information and access the desired test results. Theresultant analysis can be printed on a printer 606 or displayed on adisplay 608.

The removable carousel 114 of the pipetter-diluter device 100 acceptstest tubes 134 in an upright configuration. The tubes 134, acceptedalong the periphery of the carousel 114 in openings 124 conforming tothe cross-sections of the tubes 134, have been previously centrifuged ina conventional centrifuge device to separate, by density, specimens ofinterest contained therein. An empty dilution cup 134a corresponding toeach tube 134 is accepted in the inner ring of openings 122 conformingto the cross-sections of the cups 134a. The dilution cups 134a are usedto contain liquid for diluting the specimen removed from the specimentubes 134. The cups 134a may be conventional Krone cups with V-bottoms122.

As illustrated in FIG. 12, the carousel 114 includes a stanchion 118that supports the top plate 120, the middle plate 130 and the bottomplate 132. The cups 134a are supported by their lips 124 on the topplate 120. The test tubes 134 are supported atop the bottom plate 132 bythe middle plate 130 and top plate 132.

The carousel 114 is removably held in the trough 128 below the planarwork surface 20 of the biological fluid assay apparatus 10. The centerstanchion 118 of the carousel 114 extends slightly above the planar worksurface 20. On its lower end, the stanchion 118 telescopes over an axle119 with radially outwardly directed tabs 121 that engage slots 123 inthe inside surface of the stanchion 118.

The test tubes 134 may be adapted to hold a smaller volume, while havingsufficient height to permit definition of the layers within a specimen,through the use of the removable inserts 136. Each insert 136 includesan upper enlarged diameter funnel portion 138 that frictionally mateswith the inside wall of a test tube 134. A narrow channel 140 extendsdownwardly through the test tube 134 from the conical bottom 139 of theportion 138. In this way a conventional test tube 134, having a volumeof 6 to 7 milliliters for example, is adapted to contain a lesservolume, for example about 1 milliliter, in the insert 136, which hassubstantially the same height as the test tube 134.

A coded bar label 144 fits on the exterior facing side of the tubes 134.Therefore, the optical bar code reader 24 may positively identify allspecimens through their labels 114. The carousel 114 may be atomaticallyrotatively indexed to align successive tubes 114 with the reader 24 orto orient a tube 134 in a desired position, through the rotation of thestanchion 118 by the axle 119. the support plate 350, in turn supportedby the stanchions 352. The pivotal cover 16 encloses the centrifugerotor 300 inside the biological fluid assay apparatus 10. The cover 16can be lifted to provide access to the centrifuge rotor 300.

The pipetter-diluter device 100 includes a syringe 102 and a conduitneedle 106 in fluid communication. The needle 106 is automatically movedfrom location to location by the L-shaped rotating and reciprocating arm108. The arm 108 comprises the vertical stanchion 112 and the horizontalstanchion 110.

To prevent contamination caused by utilizing the conduit needle 106 andthe syringe 102 with different fluids, a needle wash station 116 is usedto wash the conduit needle 106 and the syringe 102 between contact withthe different fluids. The wash station 116 is located along the arc "A",shown in FIG. 1, circumscribed by the needle 106 as it pivots with thearm 108 to and from the appropriately aligned tubes 134 and cups 134a.

The reagent reservoirs 202 are located on top of the planar work surface20 and are secured by the reservoir cover 204. The reagent reservoirs202 are connected to the reagent pump 206 by the exit tubes 208. Thereagent pump 206 is enclosed in the upwardly protruding section of thetop structure 12 that lies above the planar work surface 20. The reagentpump 206 provides reagent to the outlets 210 by the tubes 212.

The electrical components associated with the centrifuge rotor 300 arelocated below the support plate 350, as shown in FIGS. 2 and 3. Thevariable speed motor 304, the stepping motor 308 and the solenoid 306are located blow the support plate 350. The variable speed motor 304 isconnected to the centrifuge rotor 300 by a series of shafts and pulleys.The stepping motor 308 is engaged and disengaged by the solenoid 306, asshown in FIG. 3. The solenoid 306 draws the movable core 322 into aninner coil (not shown) when current flows through the solenoid 306. Asthe movable core 322 is pulled through the spring 304 into the solenoid306, the lever 326 is pivoted about the pivot point 328. When the lever326 pivots about the pivot point 328, the clutch 330 is forced to engagethe pulleys 312 and 332. The pulley 312 is driven by the variable speedmotor 304 utilizing the belt 314. The pulley 332 is driven by the belt334 which is operationally associated with the stepping motor 308. Thestepping motor 308 is connected to the pulley 332 by the shaft 338 andthe pulley 336. The main shaft 310 is fixed to the centrifuge rotor 300by the mounting plate 316.

As shown in FIG. 4, the variable speed motor 304 drives the pulley 356,the belt 314, the pulley 312, the shaft 310, the mounting plate 316 andultimately the centrifuge rotor 300. The stepping motor 308 drives thestepping motor pulley 336, the belt 334, the pulley 332, the shaft 310,the mounting plate 316 and ultimately the centrifuge rotor 300.

The single use belt 400 encircles the centrifuge rotor 300 as shown inFIG. 3. The single use belt 400 is held in place by the belt supports302 located between the cuvettes 402 of the single use belt 400 whichmay be a strip folded upon itself to assume a cylindrical configuration.Generally, the single use belt 400 can be made of any material that istranslucent, transparent or pellucid to light. The belt 400 can be madeof transparent, flexible plastic such as PVC, styrene, celluloseacetate, cellulose butyrate, or any relatively inert, lighttransmissive, wettable plastic material that is compatible withbiological reagents used in the testing of biological specimens.Additionally, it is preferred that the belt 400 be made of a suitabletranslucent material that diffuses the light and provides a more uniformillumination of the sample to enhance the optical resolution.

As shown in FIGS. 6, 7 and 8 each cuvette 402 in the belt 400 is formedfrom an elongate flat strip 406 fixed to the elongate, indented strip408 shaped by vacuum forming or the like. The flat strip 406 and theindented strip 408 form the inlets 404 and the chamber 418. Oneembodiment of the present invention utilizes a single use belt 400having eighty-four cuvettes 402, for example, with seven cuvettes 402available for each series of reactions required to determine the bloodgroup of a patient. Thus, twelve patient specimens can be analyzed usinga single use belt 400 having eighty-four cuvettes 402.

The vertically aligned, convex chambers 418 smoothly extend radiallyoutwardly. Each cuvette 402 is formed in the shape of an angled,inverted test tube portion extending upwardly and outwardly from thevertical plane defined by the strip 406. Each cuvette 402 is broadlysemi-elliptical in shape when viewed from above, as shown in FIG. 7.Typically, the indented strip 408 is heat sealed to the flat strip 406.As shown in FIG. 8, a vertical apex 410 is located at the bottom of thechamber 418. The moderately inclined segment 414 increases in depth, toa radial apex 412. The segment 414 may be oriented at an angle of about30° with respect to the strip 406. The spherically shaped radial apex412, having the shape of the bottom of a conventional test tube, is themost outwardly radially extending part of the cuvette 402. After theradial apex 412, the outline of the cuvette 402 is angled sharply backtoward the strip 406 by the segment 416. Each cuvette 402 then extendsvertically upwardly along a uniformly shallow generally vertical segment420. The uniformly shallow generally vertical segment 420 in conjunctionwith the flat strip 406 form the inlet 404.

The smooth characteristics of the cuvette 402 greatly enhance theutilization of the present invention. The smoothly curved surfacesassociated with the indented strip 408 provide very little physicalresistance to the movement of fluid thereupon for enhancing thesensitivity of the biological fluid assay system 10. Thus, surfacetension is minimized, flow characteristics are enhanced and a betterqualitative determination of the characteristics of the immunologicalreaction is possible.

The centrifuge rotor 300 is comprised of a top plate 340, an annularplate 344 and a cylindrical member 342, as shown in FIG. 3. The topplate 340 is fixed at right angles to the central axis of thecylindrical member 342. The top plate 340 and the cylindrical member 342are secured to the annular plate 344. The annular plate 344 and thecylindrical member 342 have a plurality of aligned apertures 354. Theapertures 354 are approximately the width of two of the cuvettes 402 onthe belt 400. Typically, as illustrated in FIG. 4, two of the cuvettes402 are aligned directly in front of each aperture 354 when the singleuse belt 400 is engaged with the centrifuge rotor 300. The orientationof the single use belt 400 is maintained by the belt supports 402. Thebelt supports 402 abut one or more cuvettes to positionally secure thesingle use belt 400 on the centrifuge rotor 300. Thus, as illustrated inFIG. 3, when the centrifuge rotor 300 revolves, each individual cuvette402 on the single use belt 400 passes through the optical system 500 foranalysis. The centrifuge rotor 300 is steadily guided by the rotor guide346 and mating slotted member 348.

The conduit needle 106 and syringe 102 withdraw specimen from eachspecimen tube 134 for deposit either in a cuvette 402 (See FIGS. 3-8) orin a dilution cup 134a. After specimen has been introduced, the conduitneedle 106 and the syringe 102 are used to deposit diluent into thedilution cups 134a. The specimen/diluent solution is withdrawn from thedilution cups 134a by the conduit needle 106 and the syringe 102 fordeposit in a cuvette 402. The conduit needle 106 acquires access to thecuvettes 402 in the single use belt 400 by passing through the aperture18 in the cover 16. The diluent is maintained in the diluent reservoir104. The syringe 102 extracts the diluent from the reservoir 104 andpasses the diluent through the conduit needle 106 to the dilution cups134a.

The appropriate angular acceleration of each cuvette 402 by thecentrifuge rotor 300 is important. The centrifugal force causes the redcells to form a small round compact mass 92 as shown in FIGS. 9B-9E.With sufficient centrifugal force, the compact mass 92 forms at theradial apex 412 of the cuvette 402. In the illustrated embodiment, thecentrifuge rotor 300 accelerates to a velocity sufficient to impart acentrifugal force to the red cells in the specimen at a magnitude ofabout 600 G's which corresponds to approximately 2100 revolutions perminute. The centrifuge rotor 300 maintains this speed for approximately35 seconds.

When the compact mass 92 is sufficiently concentrated and compacted bythe centrifugal force, the motor 304 is automatically deenergized.Without the aid of the motor 304, the centrifuge rotor 300 decelerates.When the centrifuge rotor 300 reaches a speed at which the force ofgravity automatically equals the centrifugal force along the inclinedsegment 414 due to the rotation of the centrifuge rotor 300, thestepping motor 308 engages the shaft 310 of the centrifuge rotor 300 byutilizing the belt 334. The stepping motor 308 provides the force todrive the centrifuge rotor 300 at a speed that approximately balancesthe gravitational and centrifugal components of the force along theinclined segment 414 of the cuvette 402. Typically, the stepping motor308 drives the centrifuge rotor 300 at approximately 60 revolutions perminute or slightly less than 1G.

The optical system 500 includes the light source 502 affixed in thestationary member 506 and extending into the optical chamber 504 of themember 506, as shown in FIG. 5. The rays 509 of light emitted by thelight source 502 impinge upon the lens 508. The lens 508 focuses therays 510 through the aperture 354 in the centrifuge rotor 300 andthrough the cuvette 402. Further, the lens 508 causes the rays 510 toconverge toward the reflector 512. The rays 510 impinge upon thereflector 512 and are redirected as rays 524. The rays 524 pass into thelens box 516 through the slit 514. Once inside the lens box 516, therays 524 pass through the lens 518. The lens 518 focuses the rays 526through the transmissive surface 520 onto the linear optical detector522. The linear optical detector 522 is enclosed in the chamber 528 toprevent extraneous readings due to outside light sources. Since thelight source 502 is oriented perpendicularly to a vertical plane, onlythe vertical dimension of the specimen in each cuvette 402 is imaged.

The reagent dispensing device 200, shown in FIGS. 2, 10, and 11 includesthe stoppers 224 engaged with the reservoir cover 204 and the reservoirs202. The air tubes 222 and the exit tubes 208 protrude from the stoppers224 and go into the reagent pump 206. The reagent is pumped out of thereservoirs 202 into the reagent pump 206 and thereafter through thetubes 212 to the outlets 210. The outlets 210 are mounted on the outletsupport 214. The outlets 210 discharge reagent simultaneously into theappropriate number of cuvettes 402 aligned directly under the outlets210. The arcuate edge 222 of the outlet support 214 has the same radiusof curvature as the centrifuge rotor 300 and extends beneath the cover16. The outlet support 214 can be pivoted at the pivot point 218 by thearm 216. When pivoted at point 218, the outlets 210 are located abovethe flush reservoir 220. The reagent dispensing device 200 is cleaned byflushing solution through the device 200 into the flush reservoir 220.

The microprocessor or central processing unit (CPU) 600 controls thefirst I/O board 610, the second I/O board 612 and the third I/O board646, as shown in FIG. 15. The first I/O board 610, shown in FIG. 16,connected to the CPU 600 by the STD bus, controls the input from and theoutput to the keyboard 604, the display 608, the buzzer 609 and theprinter 606. In the present invention, a printer interface board 654 isused to transfer data between the first I/O board 610 and the printer606.

The second I/O board 612 controls the power-driver board 614 and themotor-driver board 628, as shown in FIG. 17. The power-driver board 614controls the pump assembly 618, including the air pump 615, peristalticpump 615, air valves 617, and air pressure sensor 619. The board 614also controls the gear motor component 620, including the gear motor 621and gear motor sensor 623, the solenoid 306, the cover 16 interlocksensor 624 and the reagent valve assembly 616, including the valvemotors 625, 627. The cover interlock sensor 624 insures that the cover16 is closed and provides an appropriate indication on the keyboard 604(FIG. 16) if the cover 16 is open. The air pump 613 supplies air throughthe air valves 617 and pressure sensors 619 to the air tubes 222 and theperistaltic pump 615 to control the dispensing of fluid from thedispensing device 200. The reagent valve assembly 618 includes tworotary multipart valves driven by the valve motors 625, 627. The valveposition sensors 635 enable selective dispensing from the reservoirs202. The gear motor 621 is operative to index the carousel from oneangular position to the next through the engagement of the stanchion 118by the axle 119.

As shown in FIG. 18, the motor-driver board 628 powers the clutchcontrol 632, clutch sensor 633 the power supply 634 for the steppingmotor 308, the power supply 636 for the variable speed motor 304, thelight source 502, the controls 640 for the linear optical detector 522,the heat sink 644, and the controls 642 for the stepping motor 308, andthe servo motor 630, as shown in FIG. 18. The servo motor 630 controlsthe positioning of the arm 108. The clutch sensor 633 may be an opticalsensor which detects the present clutch 330 position. The third I/Oboard 646, shown in FIG. 15, controls the video display 603. Isolators647 are used for optically isolating the various controls and motorsfrom the digital microcomputer components.

The method of the present invention is schematically described in FIG.19 and is efficiently practiced utilizing the apparatus of the presentinvention. The analysis sequence is initiated by pressing the "enter"button on the keyboard 604 (step 700). The disposable belt 400 ismanually loaded onto the centrifuge rotor 300 and secured by the beltsupports 302 (step 702). The cover 16 is lowered to enclose thecentrifuge rotor 300 and the disposable belt 400 (step 704). A bloodspecimen of approximately 1.0 milliliter is acquired from each patientand manually placed in the insert 136 of a specimen tube 134. However, a6-7 milliliter sample may be routinely used with test tube 134 withoutthe insert 136. Each specimen tube 134 has a coded bar label 144attached thereto. The specimen tubes 134 are centrifuged to separate theblood specimens into red blood cells and supernatant. The specimen tubes134 having the separated blood specimen (red cells and plasma) aremanually placed into the peripheral apertures 124 in the carousel 114.The dilution cups 134a are normally placed in the interior apertures 122in the carousel 114. The carousel 114 is loaded into the trough 128 ofthe apparatus 10 (step 706).

The analysis is begun by pressing the "start" button on the keyboard 604(step 708). The reagent dispensing device 200 simultaneously dispensesthe correct reagents into seven cuvettes 402 of the disposable belt 400(step 714). The pipetter-diluter device 100 is activated to prepare thespecimen for analysis (step 710). The operation of the pipetter-diluterdevice 100 (step 710) will be described in more detail later. During thetime the pipetter-diluter device 100 and the reagent dispensing device200 are operating, an extra carousel 114 can be prepared for the nexttest (step 712).

The reagents are held in the reagent reservoirs 202. Reagents typicallyused include antisera, anti-A, anti-B, anti-A-B and anti-D, A1 reagentred cells, A2 reagent red cells, B reagent red cells, O reagent redcells and control. In addition, Rh pheno typing antiserums such as antiC, c, E and e may be used. Other direct agglutinating antiserums such asanti-M, anti-N, anti-P and anti-K can also be used.

After the reagent dispensing device 200 has dispensed the correct amountof reagent into the cuvettes 402 and the pipetter-diluter device 100 hasprepared and transferred the specimen to the cuvettes 402, the carousel114 can be removed from the apparatus 10 and emptied (step 716). Thecentrifuge rotor 300 is then accelerated to a velocity sufficient tocompact the sample into the extreme radial apex 412 of the cuvette 402(step 720). The centrifuge rotor 300 is decelerated to a velocity wherethe gravitational attraction is slightly greater than the centrifugalforce on the specimen so that streaming of the opaque particles ispossible in negative particles (step 722). A baseline reading of theoptical characteristics of the sample in each cuvette 402 isautomatically taken using the optical system 500 (step 724) before anysignificant streaming can occur. The optical system 500 determines thevertical dimension (VI in FIG. 9B) of the opaque portion of the sample.The baseline reading is critical for determining an accurate indicationof a possible reaction. The rotating speed must be slow enough for theoptical system 500 to make the necessary measurements, while the rotor300 is spinning, on a continuous basis in a strobing fashion. Thus thespeed of rotation is limited by the capability of the optical technologyutilized.

After sufficient time has elapsed (e.g., 25 secs.) for streaming in anegative test to be completed, the optical system 500 automaticallyreads the reaction which occurred (step 730), i.e., the verticaldimension in FIG. 9C-9E, of the opaque portion of the sample isdetermined. After the final reading has been taken, the centrifuge rotor300 stops (step 732), the cover 16 can be raised (step 734), the printer606 and the display 608 are activated (step 736) and the single use belt400 can be manually unloaded. All of the data taken has been stored inthe microprocessor 600 for analysis.

The baseline reading (step 724) can be automatically compared by the CPU600 with the final reading (step 730) to determine any abnormality ormalfunction associated with the specific sample. For example, thebaseline reading may indicate that the sample had "slipped out" of theradial apex 412 of the cuvette 402. Such slippage could be interpretedas the streaming of the sample caused by agglutination rather than bythe fact that the entire opaque portion of the sample slid down inposition which would be an incorrect reading. The procedure fordetermining the baseline characteristics of the sample preventsmisreading in this and similar situations.

When a "slipped" reaction 84, shown in FIG. 6, is noted, themicroprocessor 600 requires the printer 606 or the display 608 to notethe inconsistency of the reading with a "?". A weak reaction mass 88,shown in FIG. 6, is illustrated adjacent the negative reaction mass 86.A weak reaction could be related to specific biological parameters bythe microprocessor 600 and indicated by the display 608 or the printer606.

The second vertical measurement V2, as illustrated in FIGS. 9C, 9D and9E, is compared by the microprocessor 600 with the first verticalmeasurement V1, as illustrated in FIG. 9B, by subtracting V2 from V1. Ifthe difference between V1 and V2 exceeds a preset difference asdetermined by the microprocessor 600, a negative reaction is indicated.If the difference does not exceed the preset value as determined by themicroprocessor 600, a positive reaction is indicated, as shown at 82 inFIG. 6. No agglutination (i.e. streaming) indicates a negative test andagglutination (no streaming) indicates a positive test.

It will be understood that by taking the vertical measurements V1 and V2of the compacted red blood cells of each individual sample, the presentinvention provides baselining for each sample. The utilization ofbaselining in the present invention provides a standard against whichmeasurements can be compared. Further, such baselining as illustrated inFIGS. 9A through 9E, eliminates any effects on the determination ofreactions due to variations in the volume or concentration of the opaqueparticles of the specimen. Still further, and perhaps moresignificantly, such baselining eliminates any effect in differentiatingbetween positive and negative reactions due to extraneous verticaldownward movement of the compacted red blood cells, as indicated at 84in FIG. 6. Such downward movement is known to occur and withoutbaselining would indicate a false negative reaction.

The microprocessor 600 evaluates the reaction by reading the responsesof the individual photosensitive elements (pixels) of the linear opticaldetector 522 in sequence based upon the absorbed energy. The image ofthe compacted red blood cells shadows the individual photosensitiveelements (pixels). The pixels shadowed by the projection of thecompacted red blood cells onto the linear optical detector 522 have lowenergy output. The pixels exposed directly to the light rays 509 and notshadowed by the compacted red blood cells 92 have a high energy output.The number of the low output, dark pixels 530 is directly proportionalto the length of the compacted red blood cells 92 in the cuvette 402. Ifthere are significantly more low output, dark pixels due to thestreaming red blood cells, this would indicate a negative reaction. Thestrength or weakness of the reaction would be proportional to theincrease in the number of low output, dark pixels on the linear opticaldetector 522. The magnitude of the dimension of the image or shadowproduced can be used as a comparison to determine the presence andmagnitude of a reaction between a red blood cell specimen and a reagent.A battery of such measurements is determinative of blood type.

FIG. 20 illustrates the automated sequence performed by thepipetter-diluter 100. Initially, the bar coded labels 144 on twelvetubes 134 in the carousel 114 are read by a conventional optical codereader 24 (step 800). The arm 108 is rotated to be aligned over thefirst tube 134 in the carousel 114 (step 802). The arm 108 is lowered toallow the needle 106 to engage the less dense supernatant (plasma) thatrests above the red blood cells in the tube 134 (step 804). The needle106 withdraws about 90 microliters of the supernatant (step 806). Thearm 108 is raised to disengage the carousel 114 (step 808). The arm 108is rotated to be aligned with the aperture 18 in the cover 16 over thecentrifuge rotor 300 (step 810). The arm 108 is lowered to positionitself just above a first cuvette 402 in the disposable belt 400 (step812). The pipetter-diluter device 100 dispenses about 30 microliters ofthe supernatant into the cuvette 402 (step 814). The centrifuge rotor300 is automatically indexed by the microprocessor 600 to locate anothercuvette 402 directly beneath the needle 106 (step 816). Thepipetter-diluter device 100 repeats the process of dispensing about 30microliters of the supernatant into three cuvettes 402.

After the three cuvettes 402 that already contained the requiredreagents (See FIG. 19, step 714) have received about 30 microliters eachof supernatant, the arm 108 is lifted to disengage from the cover 16.The arm 108 is rotated to again align the needle 106 with the first tube134 in the carousel 114 (step 820). The arm 108 is lowered into the tube134 through the supernatant to engage the red blood cells in the lowerextremity of the tube 134 (step 822). The pipetter-diluter device 100extracts about 20 microliters of the red blood cells from the lowerextremity of the tube 134 with the needle 106 (step 824). The arm 108 islifted to disengage the carousel 114 (step 826). The arm 108 is rotatedto align the needle 106 with a corresponding dilution cup 134a. Thedilution cup 134a is used to dilute the red blood cells extracted fromthe outer tube 134 (step 828). The arm 108 is lowered into the dilutioncup 134a (step 830).

The pipetter-diluter device 100 utilizing the syringe 102 and the needle106 dispenses the red blood cells and a diluent into the dilution cup134a (step 832). The dispersion of the red blood cells and the diluentinto the inner cup 134a is sufficient to cause adequate mixing of thered blood cells and the diluent. Typically, for human blood, thepipetter-diluter device 100 mixes about 465 microliters of the diluentwith about 20 microliters of the red blood cells to form a cellsuspension having approximately a 3.5% concentration. The 3.5% cellsuspension provides optimal reading characteristics for the presentlypreferred embodiment of the present invention.

The needle 106 extracts about 120 microliters of the 3.5% cellsuspension from the inner cup 134a (step 834). The arm 108 lifts todisengage from the carousel 114 (step 836). The arm 108 rotates to againbe aligned with the aperture 18 in the cover 16 (step 838). The needle106 is lowered into the aperture 18 to be aligned above the fourthcuvette 402 which already contains an appropriate reagent (step 840).The pipetter-diluter device 100 dispenses approximately 30 microlitersof the 3.5% cell suspension into the cuvette 402 with the needle 106(step 842). The centrifuge rotor 300 automatically indexes to the nextappropriate cuvette (step 844). The dispense-index sequence is continueduntil a total of four cuvettes containing an appropriate reagent haveaccepted the 30 microliters of the 3.5% cell suspension. Thereafter, thearm 108 is lifted to disengage from the cover 16 (step 846). Thecentrifuge rotor 300 is indexed to the next appropriate cuvette 402(step 848). The carousel 114 is indexed to align the next tube 134 (step852) with the path of the needle 106 and the cycle begins again (step802) and is repeated for all twelve specimens in each tube 134.

The needle 106 is washed between patients in the wash station 116 bypurging with a cell suspending solution contained in the diluentreservoir 104. The red cell suspending solution may comprise anhydrousdextrose, sodium chloride, sodium citrate, citric acid, deionized water,chloramphenicol, neomycin sulfate, and imosine.

When the fluid assay system 10 is not in use overnight the reagentreservoirs 202 may be removed and replaced with bottles containingsterile water. The reagent dispensing device 200 flushes the lines withthe water to leave the water in the reagent dispensing device 200 whenthe device is not being used. Similarly, the reagent dispensing device200 can be flushed with a bleach solution and water at periodicintervals to clean the interior components of the reagent dispensingdevice 200.

The number of cuvettes 402 to receive supernatant, the number ofreagents utlized, and the number of specimens handled by the system 10may be preprogrammed by an operator. Further, the volume of specimenwithdrawn from the carousel may be varied.

The method of the present invention can be utilized with any specimenthat can be separated by density and contains opaque material that hasphysical reactions with a specific reagent. It is preferable that thereactions cause the opaque portion of the specimen to become more fluidor less fluid depending upon the characteristics of the specimen.

Additional advantages and modifications of the present invention willreadily occur to those skilled in the art. The invention in its broaderaspects is therefore not limited to the specific details, representativeapparatus, and the illustrative examples shown and described herein.Accordingly, departures may be made from the detail without departingfrom the spirit or scope of the disclosed general inventive concept.

What is claimed is:
 1. An apparatus for determining the reactivecharacteristics of a biological mixture of a specimen and a reagentcomprising:(a) a centrifuge rotor; (b) an energy transmissive, flexiblebelt formable into a cylinder and removably mounted for movement inunison with said centrifuge rotor, said belt including a plurality ofspecimen containers, said specimen containers each including a flatelement having an inner surface, an outer surface, an upper surface anda lower end, and an indented element having an inner surface, an outersurface, an upper end and a lower end, and having at least one smooth,outwardly, radially extending and vertically oriented indentation formedtherein, the indentation vertically gradually increasing in an outwardlyextending radial depth along a moderatley inclined slanting segment to asmoothly curved radial apex then abruptly decreasing in radial depthalong a severely inclined segment and thereafter extending upwardlyalong a uniformly shallow generally vertical segment to the upper end ofsaid indented element, said flat element and said indented element beingconnected together with the inner surface of the flat element in contactwith the inner surface of said indented element, said open upper end ofsaid indented element forming an opening for filling said specimencontainers; and (c) a detection system for irradiating the plurality ofspecimens contained within said belt and for optically measuring thereactive characteristics of the specimens.
 2. An apparatus as describedin claim 1 including means for automatically making a first reading of alinear dimension of specimens contained in said belt, means forautomatically making a second reading, after the lapse of a period oftime, of a linear dimension of specimens contained in said belt andmeans for automatically comparing said first and second readings.
 3. Anapparatus as described in claim 1 wherein said centrifuge rotorcomprises:(a) an essentially horizontal circular planar member; (b) acylindrical member having an upper end, a lower end, an inner surfaceand an outer surface; and (c) an annular member fixedly securing saidplanar member perpendicular to the central axis of said cylindricalmember said annular member disposed at an acute angle with the plane ofthe planar member and the central axis of said cylindrical member, aplurality of recesses formed in said annular member and said upper endof said cylindrical member said recesses adapted to align with saidindentations in said indented element.
 4. An apparatus as described inclaim 3 further comprising a plurality of supports fixedly secured tothe lower end of said cylindrical member and extending upwardly forremovably securing said energy transmissive belt to said centrifugerotor.
 5. An apparatus as described in claim 1 including a housingadapted to contain said centrifuge rotor and said detection system, saidapparatus further including a pipetter-diluter device mounted in saidhousing, said pipetter-diluter device comprising:(a) a syringe forwithdrawing fluid from a reservoir, holding fluid and discharging fluid,(b) a conduit needle operationally associated with said syringe forinsertion into the fluid to permit said syringe to withdraw or dischargethe fluid, (c) a carousel, and (d) a plurality of reservoirs interposedin said carousel from which said conduit needle and said syringe canwithdraw fluid or into which said conduit needle and said syringe candischarge fluid.
 6. An apparatus as described in claim 5 wherein saidcarousel comprises:(a) a stanchion, and (b) a plurality of flat circularplates fixedly engaged with and perpendicular to said stanchion foraccepting and for securing said plurality of reservoirs, said pluralityof flat circular plates comprising a top plate having aperturesconforming to the associated cross-sections of and securing the relativeposition of said reservoirs, a bottom plate for supporting saidreservoirs, and an intermediate plate having apertures conforming to theassociated cross-sections of and securing the relative position of saidreservoirs.
 7. An apparatus as described in claim 1 wherein saidcarousel includes a plurality of receptacles, said receptacles hayingcoded bar labels on said receptacles, said apparatus further including acoded bar label reader arranged to read the labels on said receptacles,said apparatus including means for indexing said carousel past saidreader.
 8. An apparatus as described in claim 7 including means forautomatically inserting reagents and specimens into said plurality ofcontainers in the proper amounts.
 9. An apparatus for determining thereactive characteristics of a biological mixture of a specimen and areagent comprising:a centrifuge including receptacles for a plurality ofseparate specimen and reagent containing samples; a device forautomatically measuring at least one dimension of each of said samplesin said receptacles while said centrifuge is rotating; and a comparatorfor comparing, for each sample, a representation of a first measurementof a dimension made by said device with a representation of a subsequentmeasurement of a corresponding dimension made by said device.
 10. Anapparatus as described in claim 9 wherein said centrifuge includes aremovable flexible belt having said receptacles formed in said belt. 11.An apparatus as described in claim 9 wherein said device automaticallymakes a subsequent measurement of a vertical dimension of each of saidsamples after the expiration of a predetermined time period followingthe first measurement.
 12. An apparatus as described in claim 9 wereinsaid comparator is adapted to compare the difference between said firstand subsequent measurements with a predetermined difference indicativeof a reactive characteristic between said specimen and reagent.
 13. Aspecimen holder for a centrifuge apparatus adapted to determine thereactive characteristics of a biological mixture of a specimen and areagent, said holder comprising an energy trnasmissive, flexible,disposable belt having a pair of opposed sides and side edges, said beltbeing formable into a cylinder and removably mountable for movement inunison with a centrifuge apparatus, said belt having a plurality ofspecimen containers formed integrally in said belt and spaced at regularintervals along the length of said belt, said containers extendingoutwardly from one of said sides of said belt, and the other of saidsides of said belt being flat, each of said containers having a fillingopening near the same side edge of said belt and each of said containershaving a first face generally conincident with the plane of said beltand an opposite face, said opposite face including a smoothly curvedradial apex spaced outwardly from the plane of said belt and a portionwhich is inclined with respect to said plane of said belt and whichconnects said apex to the remainder of said belt, said portionvertically gradually increasing in an outwardly extending radial depthalong a moderately inclined slanting segment to said apex then abruptlydecreasing in radial depth along a severely inclined segment andthereafter extending upwardly along a uniformly shallow generallyvertical segment ot said filling opening.
 14. The specimen holder ofclaim 13 wherein said holder is optically transparent.
 15. The specimenholder of claim 14 wherein each of said sides is formed of a sheet ofplastic film, said sides being connected to one another.